Microorganisms capable of metabolizing cholesterol are potential sources of enzymes useful in an enzymatic assay of cholestrol in complex mixtures such as blood serum, etc. This is particularly so if the microorganisms can use cholesterol as a sole carbon source, for in this assay process cholesterol must be degraded by oxidative enzymes.
Stadtman, T. C., Methods in Enzymology, Vol. 1; Colowick, S. P., and Kaplan, N. O., Eds. Academic Press, N.Y., 1955, p. 678; and Stadtman, T. C., Cherkes, A., and Anfinsen, J., Biol. Chem., 206, 510 (1954), reported the preliminary purification of an enzyme from Nocardia cholesterolicum, an organism originally isolated by Schatz et al (Schatz, A., Savard, K., and Pintner, I. J., J. Bacteriol., 58, 117-125 (1949). Stadtman's enzyme, "cholesterol dehydroenase", was purified sufficiently for use in a cholesterol assay based on the measurement of the increase in absorbance at 250 nm. owing to the formation of cholest-4-en-3-one. Since, as we have now determined, the direct acceptor of cholesterol electrons in this oxidation is oxygen, the enzyme should properly be called cholesterol oxidase according to current convention.
The bacterial strains described by Stadtman when cultured as described in the aforementioned references produce very low enzyme levels which are not practical for commercial operations. These levels are so low that commercial production of purified enzyme is a very remote possibility.
Goodhue et al., in U.S. Pat. No. 3,909,359 issued September 30, 1975, describe an improved method for the production of the Stadtman cholesterol oxidase, which method comprises the steps of:
a. growing the bacterium Nocardia cholsterolicum species NRRL 5767 or NRRL 5768 in a medium in which cholesterol or a suitable derivative thereof serves as an auxiliary source of carbon and PA1 b. isolating from said medium cell-free extract containing the active enzyme.
The method described by Goodhue et al is greatly improved over the original synthesis described by Stadtman and can be said to render the process commercially practical.
German OLS 2,246,695 published Mar. 29, 1973, describes a method for isolating a cholesterol oxidase enzyme produced by a culture of Nocardia microoorganism identified as NRRL 5635 and NRRL 5636. According to the method described therein, the harvested cells are treated with a nonionic surfactant and stirred at room temperature to release a large proportion of the enzyme from the cells into the supernatant, thereby eliminating the need for involved cell extraction and isolation techniques. From the data reported, we have calculated the extraction yields enzyme activity on the order of about 40 to 160 U/liter.
Reese, E. T., and Maquire, A., in "Surfactants as Stimulants of Enzyme Production by Microorganisms", Applied Microbiology, February, 1969, pp. 242-245, describe the observation that the addition of sorbitan polyoxyethylene monoleate (Tween 80 from Atlas Chemical Co., Wilmington, Delaware) and other nonionic surfactants to fungal cultures which normally produce extracellular enzymes results in a marked increase in enzyme yield.
British Patent 1,385,319 describes the production of cholesterol oxidase from Nocardia species NRRL 5635 and NRRL 5636. The fermentation is conducted in a growth medium containing 20 g./liter of yeast extract. During the fermentation, cholesterol is slowly added to the medium, preferably in the form of a dispersion containing a nonionic surfactant. Cholesterol is added in quantities totaling up to 1.2 g./liter of medium during this addition. The total concentration of nonionic surfactant added in this manner is minute.
Republic of South Africa Patent 73/3259 describes the production of cholesterol oxidase using Proactinomyces erythropolis NBC 9158, ATC 17895, ATCC 4277 and Nocardia formica ATCC 14811. The fermentation is conducted in a peptone-containing mineral salt medium and, when the logarithmic growth phase is reached, cholesterol in the form of an aqueous suspension is slowly added to the medium in proportion to the growth of the microorganism such that the cholesterol added totals 1 to 20 g./liter of medium. A small amount of cholesterol (0.05% in Example 2) may be added to the medium initially. Increase in the yield of cholesterol oxidase activity is obtained by adding yeast extract to the cholesterol suspension as an emulsifying agent in an amount of 0.02 to 1% by weight of the cholesterol suspension. It can be seen that only a very small amount of yeast extract is added to the medium in this manner. No surfactant is used during the fermentation.